NusG/Spt5 proteins are the only known universally conserved transcriptional regulators that coevolved with RNA polymerase since the last universal common ancestor. The best studied members of this family are Escherichia coli NusG, an essential housekeeping protein that acts together with the transcription termination factor Rho to silence foreign DNA and antisense transcription, and its paralog RfaH, a non-essential protein required for expression of a few horizontally-acquired operons that contain an ops sequence in their leader regions. In our previous work, we established RfaH as a model to elucidate the mechanism of transcription processivity conferred by NusG proteins. However, recent structural studies by us and others propose paradigm-shifting models for how NusG proteins function. The new structures revealed unexpected interactions and suggested that NusG proteins may promote elongation by facilitating translocation, inhibiting backtracking and swiveling, chaperoning the nascent RNA, and constraining the flexible nontemplate DNA strand. These hypotheses require extensive validation that we propose to carry out with RfaH in Aim 1. RfaH activates gene expression in part by silencing Rho, which imposes strong polarity in RfaH-controlled operons, yet these operons lack canonical Ro recognition sequences. Using a genetic selection for suppressors of ?rfaH, we identified alterations in a flexible connector region of Rho that we propose disrupt an allosteric signal required for termination. In Aim 2, we will study the role of the connector region and NusG in potentiating Rho action at suboptimal RNAs. Activation of gene expression by RfaH is thought to be mediated by recruitment of ribosome to RfaH-target RNAs that are missing Shine-Dalgarno motifs. In Aim 3, we will determine where the ribosome is recruited to the transcribing RNA polymerase and whether it scans after loading on mRNA. We will also test if RfaH couples transcription and translation and will look for new factors affecting translation of RfaH-dependent operons.